Transformation of hematopoietic cells and activation of JAK2-V617F by IL-27R, a component of a heterodimeric type I receptor

Anuradha Pradhan, Que T. Lambert, and Gary W. Reuther*

Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612

Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved October 5, 2007 (received for review March 14, 2007) From a patient with acute myeloid leukemia (AML), we have shown to be leukemogenic in mice (9), further validating our identified IL-27Ra (also known as TCCR and WSX1) as a gene whose approach to identify genes that contribute to leukemia formation. expression can induce the transformation of hematopoietic cells. In this study, we have used a functional genetic screen to IL-27Ra (IL-27R) is a type I that functions as the identify genes that contribute to myeloid cell transformation and binding component of the receptor for IL-27 and functions perhaps AML formation. Here, we describe our identification of with the (gp130) coreceptor to induce signal the ligand binding component of the receptor for IL-27 (IL- transduction in response to IL-27. We show that IL-27R is expressed 27R), as a unique transforming gene product. We show that on the cell surface of the leukemic cells of AML patients. 32D IL-27R is expressed on the cell surface of leukemic cells of AML myeloid cells transformed by IL-27R contain elevated levels of patients and demonstrate that IL-27R can transform hemato- activated forms of various signaling proteins, including JAK1, JAK2, poietic cells in a JAK-dependent manner. We also show that STAT1, STAT3, STAT5, and ERK1/2. Inhibition of JAK family proteins IL-27R activates JAK2-V617F. Previously, only homodimeric induces cell cycle arrest and apoptosis in these cells, suggesting the type I cytokine receptors have been reported to activate this transforming properties of IL-27R depend on the activity of JAK important JAK2 mutant. Our data indicate that a single com- family members. IL-27R also transforms BaF3 cells to cytokine ponent of a heterodimeric cytokine receptor has the ability to independence. Because BaF3 cells lack expression of gp130, this aberrantly activate signaling pathways in hematopoietic cells. finding suggests that IL-27R-mediated transformation of hemato- Thus, it is possible that components of heterodimeric cytokine poietic cells is gp130-independent. Finally, we show that IL-27R can receptors may play a role in myeloid diseases either through functionally replace a homodimeric in the aberrant expression or activating mutations. activation of JAK2-V617F, a critical JAK2 mutation in various myeloproliferative disorders (MPDs). Our data demonstrate that Results IL-27R possesses hematopoietic cell-transforming properties and Functional Genetic Screens to Identify Myeloid Cell Oncogenes Un- suggest that, analogous to homodimeric type I cytokine receptors, cover Unique Transforming Properties of IL-27R. To identify onco- single-chain components of heterodimeric receptors can also en- genes involved in AML, or perhaps other myeloid disorders, we hance the activation of JAK2-V617F. Therefore, such receptors may developed functional genetic screens in myeloid cells. We used play unappreciated roles in MPDs. 32D myeloid progenitor cells, which depend on IL-3 for growth and viability (10). When 32D cells are cultured in the absence of acute myeloid leukemia ͉ myeloproliferative disease ͉ oncogene ͉ IL-3, they undergo cell cycle arrest and apoptosis (11). These signaling cells have served as a model cell system for studying the transforming properties of various leukemia-associated onco- cute myeloid leukemia (AML) is a disease of the myeloid genes, such as Bcr/Abl and Flt3, among others (12, 13). For our Acompartment of the hematopoietic system characterized by approach, we used cDNA representing the genes expressed in an accumulation of undifferentiated blast cells in the peripheral the leukemic cells of patients with AML. This cDNA was cloned blood and bone marrow. AML is caused by multiple genetic and into a retroviral vector (pEYK3.1; a gift of George Daley, epigenetic changes that result in stimulation of mitogenic signals Harvard Medical School, Children’s Hospital, Boston) designed and deregulation of apoptosis and differentiation. It has been for efficient delivery and proviral recovery (14). We generated proposed that mutations in two different classes of oncogenes retrovirus containing cDNA libraries from AML patients and are required to induce AML. Mutations in class I oncogenes used this virus to infect 32D cells that exogenously express B-cell (e.g., Ras, Flt3) result in stimulation of proliferative and cell CLL/lymphoma 2 (Bcl2). survival signals, whereas mutations in class II oncogenes (e.g., We used 32D/Bcl2 cells for multiple reasons. First, removal of AML1-Eto, PML-RAR␣) lead to inhibition of differentiation and IL-3 leads to a very rapid apoptotic cell death of 32D cells, and subsequent cell death by apoptosis (1–3). Although many mu- our screens of parental 32D cells resulted in no cytokine- tations are recurrently found in AML patients, it is believed that independent isolates. Second, oncogenic expression in 32D cells additional mutations in AML exist and have yet to be identified needs to elicit an antiapoptotic and a mitogenic signal to (1, 2, 4–6). Even with a large knowledge base about the causative transform these cells to cytokine independence. Third, it is genetics of AML, a more complete understanding of the mo- lecular players is needed to identify targets for future therapeutic treatment for this disease. Author contributions: A.P. and G.W.R. designed research; A.P., Q.T.L., and G.W.R. per- We have previously performed functional genetic screens to formed research; A.P., Q.T.L., and G.W.R. analyzed data; and G.W.R. wrote the paper. identify oncogenes in AML by using an efficient retroviral The authors declare no conflict of interest. delivery, expression, and cDNA recovery system (7, 8). Using This article is a PNAS Direct Submission. this approach, we identified an activating deletion mutation in *To whom correspondence should be addressed. E-mail: gary.reuther@moffitt.org. the TrkA tyrosine kinase in a patient with AML (7). This This article contains supporting information online at www.pnas.org/cgi/content/full/ discovery provided evidence that TrkA may play a role in 0702388104/DC1. leukemogenesis. The deletion mutation we identified has been © 2007 by The National Academy of Sciences of the USA

18502–18507 ͉ PNAS ͉ November 20, 2007 ͉ vol. 104 ͉ no. 47 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702388104 Downloaded by guest on September 28, 2021 Fig. 1. IL-27R transforms 32D cells to IL-3 independence. (A) 32D cells stably expressing IL-27R (F) or a control vector (■) were cultured in the absence of IL-3 starting on day 0. The total number of viable cells was determined at each time point by trypan blue exclusion. The dotted line represents the total number of viable cells going below the limit of detection of a hemacytometer and to zero. (B) Cell lysates of 32D cells stably expressing a control vector (lane 1) or IL-27R (lane 2) were immunoblotted with anti-IL-27R antibodies. (C) Antibodies that recognize the indicated proteins were used to immunoblot lysates from control 32D cells expressing an empty vector (lanes 1 and 3) or IL-27R (lanes 2 and 4) after1hofgrowth factor starvation. (D) Bone marrow mononuclear cells from normal and AML patients were analyzed by flow cytometry using an Alexa Fluor-647-conjugated anti-IL-27R antibody. The percentage of IL-27R-positive cells of each patient sample (represented by F) is plotted.

believed that transformation of myeloid cells in AML is caused STAT1, STAT3, and STAT5 are frequently activated by leuke- by mutations that activate cooperating signaling pathways (1–3). mogenic oncogenes and are activated in AML (22, 23). JAK Culturing 32D cells without IL-3 resulted in cell death of the proteins, in particular JAK2, are mutationally activated in entire culture in a little over 2 days. However, expression of Bcl2 myeloproliferative disorders (MPDs) (24–28) and a small frac- led to enhanced cell survival in the absence of IL-3 without tion of AML (29, 30). Therefore, we analyzed the activation state inducing cell growth (data not shown), which is in agreement of these proteins in 32D cells transformed by IL-27R and with previous studies (15, 16). Two days after infection, 32D/Bcl2 determined that these cells have increased phosphorylated cells were plated in medium lacking IL-3 to identify IL-3- STAT1, STAT5, JAK1, and JAK2 compared with vector control independent transformants. Using a cDNA library from an cells (Fig. 1C). STAT3 is also activated in 32D cells transformed AML patient who exhibited AML of FAB subtype M5b, a by IL-27R (see Fig. 2E). Because transformation of 32D cells monocytic leukemia with 93% blast cells and normal cytogenet- involves mitogenic signaling and inhibition of apoptosis, we also ics, we isolated a cDNA representing a WT IL-27Ra (TCCR, determined that ERK1/2 is activated in these cells (Fig. 1C). WSX-1) (17) gene from multiple independent isolates of IL-3- These data indicate that IL-27R-mediated transformation of independent cells. For simplicity, we will refer to this gene and 32D cells is associated with activation of JAK/STAT and ERK protein as IL-27R. pathways. In addition to STATs, ERKs are commonly activated in AML cells (31). Thus, transformation by IL-27R results in

Transformation of 32D cells by IL-27R. Expression of IL-27R in activation of pathways that are frequently activated in AML. CELL BIOLOGY 32D/Bcl2 cells is sufficient to transform these cells to IL-3 Activation of transforming signals by IL-27R is not associated independence (data not shown). Interestingly, IL-27R also trans- with the autocrine production of factors that stimulate cell forms parental 32D cells to cytokine independence (Fig. 1A). growth or survival (data not shown). In addition, a neutralizing These results suggest that in the context of the expression of an antibody to IL-27 did not affect the growth or viability of cells entire library of cDNAs, expression of Bcl2 in 32D cells during transformed by IL-27R [supporting information (SI) Fig. 6]. our initial screen likely sensitized these cells to transformation to IL-3 independence. Immunoblot analysis confirmed expres- Expression of IL-27R on the Cell Surface of AML Cells. Because we sion of IL-27R in these cells (Fig. 1B). The two protein bands of identified IL-27R as a transforming gene from the leukemic cells IL-27R are the result of glycosylation (data not shown). This of an AML patient, we wanted to determine whether this gene finding is consistent with the presence of multiple glycosylation was commonly expressed in AML patients. Using flow cytom- sites in the extracellular region of the receptor (17). etry, we detected IL-27R on the cell surface of AML cells. Eight IL-27R is a type I cytokine receptor that functions as the of 13 AML bone marrow samples tested had 2.5- to 10-fold more ligand binding component of the receptor for IL-27 (18, 19). cells expressing IL-27R (ranging from 0.33% to 84.6%) than the Signaling induced by IL-27 activates JAK and STAT proteins, average observed in normal bone marrow cells (ranging from including JAK1, JAK2, Tyk2, STAT1, STAT2, STAT3, STAT4, 6.3% to 12.8%) (Fig. 1D and SI Fig. 7). This finding suggests and STAT5 in various cell types (20, 21). There is clear evidence IL-27R expression is retained in many AML patients. In all that JAK/STAT proteins are activated in myeloid disorders. normal and AML samples tested, essentially all IL-27R-positive

Pradhan et al. PNAS ͉ November 20, 2007 ͉ vol. 104 ͉ no. 47 ͉ 18503 Downloaded by guest on September 28, 2021 Fig. 2. Transformation of 32D cells by IL-27R requires JAK family kinase activity. 32D cells transformed to cytokine independence by IL-27R were cultured in the presence of 0.1% DMSO [F (A) and filled bars (C)] or 0.5 ␮M JAK inhibitor I [E (A) and empty bars (C)] on day 0. (A) The total number of viable cells for each treatment was determined daily by trypan blue exclusion. Error bars indicate standard deviation within a representative experiment. (B) After 24 h of JAK inhibitor I treatment, the DNA content present in each phase of the cell cycle was determined by propidium iodide staining and flow cytometry. The percentage of cells that were in G1, S, and G2 are plotted. Error bars indicate standard deviation of triplicate samples of a representative experiment. (C) Cell viability was determined by trypan blue exclusion. Error bars indicate standard deviation within a representative experiment. (D) After 24 h of JAK inhibitor I treatment, apoptosis was determined by annexin V binding. Error bars indicate standard deviation of triplicates within a representative experiment. (E) 32D cells transformed by IL-27R were starved of serum/IL-3 for3hinthepresence of 0.1% DMSO (lane 2), 0.5 ␮M (lane 3) or 2 ␮M (lane 4) JAK inhibitor I. Control 32D cells starved of serum/IL-3 in the presence of 0.1% DMSO for3hisshown in lane 1. Cell lysates were immunoblotted with antibodies that recognize the indicated proteins. Similar results were obtained with independently derived cell lines.

cells were CD33-positive, suggesting IL-27R is expressed in cells IL-27R Transforms BaF3 Cells to Cytokine-Independent Growth. IL- of the myeloid lineage in the bone marrow (SI Fig. 7 and data 27R requires the glycoprotein 130 (gp130) coreceptor to signal not shown). in response to IL-27 stimulation (18). BaF3 cells are IL-3- dependent pro-B cells that lack gp130 expression (18, 36). IL-27R Requires JAK Activity to Transform Myeloid Cells. We ad- Through gp130 reconstitution studies, these cells have been used dressed the role of JAK family kinases in transformation by to show the requirement for gp130 to induce cell signaling in IL-27R by culturing 32D/IL-27R cells with the pan-JAK inhib- response to IL-27 (18). To test whether gp130 is required for itor, JAK inhibitor I (32). In the presence of this inhibitor the growth of these cells ceases (Fig. 2A). JAK inhibitor I induces aG1 andaG2 cell cycle arrest and a dramatic decrease in the number of cells in S-phase after 24 h of treatment (Fig. 2B). Cell viability also decreases in the presence of the inhibitor (Fig. 2C), because of the induction of apoptosis as determined by annexin V binding to the cells after 24 h of treatment with inhibitor (Fig. 2D). JAK inhibitor I not only blocks STAT1, STAT3, and STAT5 phosphorylation induced by IL-27R, but also prevents ERK1/2 activation (Fig. 2E). These experiments suggest the kinase activity of JAK family members is required for IL-27R-mediated cell growth and inhibition of apoptosis and that JAK activation is required for downstream activation of STATs and ERK1/2.

IL-27R Requires Its Box 1 Motif to Transform 32D Cells. Within the cytoplasmic region of IL-27R is a Box 1 motif (17). This motif is often found in cytokine receptors and functions as an inter- action motif with JAK proteins (33, 34). IL-27R has been shown to interact with JAK1 (35), although JAK2 is also activated after IL-27 stimulation of cells (20). Mutation of conserved prolines in Box 1 motifs generates a motif that is severely impaired in its ability to bind to JAK proteins (34). Therefore, we mutated these Fig. 3. IL-27R requires a functional Box 1 motif to transform 32D cells to residues within the IL-27R Box 1 motif to alanine and expressed cytokine independence. (A) 32D cells were infected with retrovirus encoding this mutant IL-27R in 32D cells (Fig. 3A). Removal of IL-3 from an empty vector (lane 1), WT IL-27R (lane 2), or IL-27R containing a mutant Box the growth medium of these cells results in complete cell death 1 motif (lane 3). After drug selection, cell lysates were immunoblotted for of the culture in a time course identical to cells expressing a IL-27R expression. (B) 32D cells stably expressing control vector (■), WT IL-27R F E B ( ), or IL-27R-Box1mt (Box1mt) ( ) were washed of IL-3 and cultured in RPMI control vector (Fig. 3 ). Thus, IL-27R requires its Box 1 motif medium 1640/10% FBS in the absence of IL-3 starting on day 0. The total to transform myeloid cells. Collectively, our data suggest IL- number of viable cells was determined at each time point by trypan blue 27R-mediated transformation requires receptor-mediated acti- exclusion. The dotted line represents the total number of viable cells going vation of JAK family kinases. below the limit of detection of a hemacytometer and to zero.

18504 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702388104 Pradhan et al. Downloaded by guest on September 28, 2021 Fig. 4. IL-27R transforms IL-3-dependent BaF3 cells to cytokine indepen- dence. (A) BaF3 cells infected with control vector retrovirus or retrovirus designed to express IL-27R were washed of IL-3 and plated in RPMI medium 1640/10% FBS on day 0. The total number of viable cells expressing control vector (■) or IL-27R (F) was determined at each time point by trypan blue exclusion. The dashed line represents the total viable number of cells going below the limit of detection of a hemacytometer and to zero. (B) RT-PCR Fig. 5. Activation of JAK2-V617F by IL-27R. (A) 293T cells were transfected analysis for gp130 expression in WEHI-3B cells (as a positive control), BaF3 cells, with control vector (lanes 1 and 2), WT IL-27R (lanes 3 and 4), and IL-27R- and 32D cells transformed by IL-27R. RT-PCR for STAT5 served as a control for Box1mt (lanes 5 and 6) along with WT JAK2 (lanes 1, 3, and 5) or JAK2-V617F cDNA synthesis. (lanes 2, 4, and 6). Forty-eight hours after transfection, cells were lysed and analyzed by immunoblotting with antibodies against the indicated proteins. The combination of IL-27R and JAK2-V617F led to high levels of activated JAK2 IL-27R-mediated transformation of hematopoietic cells, we and STAT5 (lane 4). IL-27R-Box1mt was impaired in its ability to activate expressed IL-27R in BaF3 cells and cultured these cells in the JAK2-V617F and STAT5 (lane 6). (B) 293T cells were transfected with control absence of IL-3. Like 32D cells, BaF3 cells are transformed to vector (lanes 1 and 2) and IL-27R (lanes 3–6) along with JAK2 (WT, lanes 1 and cytokine independence by IL-27R (Fig. 4A), which suggests that 3), JAK2-V617F (VF, lanes 2 and 4–6), gp130 siRNA (lane 5), and control nonsilencing siRNA (lane 6). Forty-eight hours after transfection, cell lysates IL-27R does not require gp130 expression to elicit a transform- were analyzed by immunoblotting with antibodies that recognize the pro- ing signal in cells and also indicates that the transforming activity teins indicated. Depletion of gp130 did not affect the ability of IL-27R to of IL-27R is not limited to 32D myeloid cells. RT-PCR analyses activate JAK2-V617F and STAT5 (lane 5). confirmed the lack of gp130 expression in BaF3 cells (Fig. 4B). Also, we could not detect gp130 expression in 32D cells trans- formed by IL-27R, suggesting that gp130 is not required for receptors and therefore be able to activate JAK2-V617F (37). transformation of these cells and is not up-regulated to facilitate We found IL-27R activates JAK2-V617F, and its downstream IL-27R-mediated signaling during transformation (Fig. 4B). target STAT5, in 293T cells (Fig. 5A). Expression of IL-27R Analyses of signaling pathways in BaF3 cells transformed by along with WT JAK2 did not lead to activation of JAK2 or IL-27R indicate similar JAK/STAT and ERK pathways are STAT5. IL-27R containing a mutated JAK-binding Box 1 motif activated as in 32D cells transformed by IL-27R (SI Fig. 8). (as in Fig. 3) was impaired in activation of JAK2-V617F and Finally, whereas IL-27R-mediated transformation is gp130- STAT5 compared with WT IL-27R (Fig. 5A). Because 293T cells independent, our observations still support the paradigm that express gp130, it is possible activation of JAK2-V617F by IL-27R IL-27-mediated signaling requires gp130 (18). IL-27-induced requires gp130. However, depletion of gp130 by siRNA had no signaling (as measured by STAT1 phosphorylation) is not ob- effect on IL-27R-mediated activation of JAK2-V617F and served in IL-27R-expressing 32D or BaF3 cells, which lack STAT5, demonstrating this effect is gp130-independent (Fig. gp130, but is seen in IL-27R-expressing 293T cells, which express 5B). Although we have not been able to coimmunoprecipitate CELL BIOLOGY gp130. siRNA depletion of gp130 in 293T cells inhibits IL-27- IL-27R and JAK2 to demonstrate a presumed interaction, we are induced phosphorylation of STAT1 (SI Fig. 9). able to detect a complex formation between IL-27R and JAK2, as well as JAK1, in vitro (SI Fig. 10), suggesting IL-27R may IL-27R Activates JAK2-V617F. Homodimeric type I cytokine recep- tors have been shown to be required for the full activation of complex with JAK2 in cells. Together, our data suggest that JAK2-V617F (37), a JAK2 mutant found in a variety of MPDs IL-27R can function in an analogous manner as homodimeric and AML (24–30, 38). It is believed homodimeric cytokine type I receptors to activate JAK2-V617F. receptors (e.g., EpoR) provide a scaffold upon which JAK2- Discussion V617F proteins can bind and become activated by transphos- phorylation (37). Such activation of mutant JAK2 is independent In an effort to identify genes that contribute to myeloid cell of ligand for the receptor. IL-27R is a type I cytokine receptor transformation, we used functional genetic screens of genes that normally functions as a heterodimeric partner with gp130 expressed in the leukemic cells of AML patients. Through our (18, 39). However, the ability of IL-27R to transform BaF3 and approach, we uncovered unique cell-transforming properties of 32D cells, which lack gp130 expression, suggests IL-27R does not IL-27R, the ligand-binding component of the receptor for IL-27 require gp130 to transform cells. Because the transforming (18). Importantly, we have determined IL-27R is frequently properties of IL-27R do not depend on its heterodimeric partner expressed on the cell surface of a greater number of bone gp130, we thought IL-27Rs could function as homodimeric marrow cells of AML patients than of cells of normal bone

Pradhan et al. PNAS ͉ November 20, 2007 ͉ vol. 104 ͉ no. 47 ͉ 18505 Downloaded by guest on September 28, 2021 marrow, suggesting it may play a role in leukemogenesis of signaling pathways in myeloid disorders and, like TpoR, such (Fig. 1D). receptors may contribute to JAK2-V617F-negative MPDs. This IL-27R is a member of the IL-6/IL-12 receptor family (40). A contribution may be through altered expression or mutation of heterodimeric receptor complex of IL-27R/gp130 is required to the receptor and can be investigated further by performing activate signaling pathways in response to IL-27 stimulation of sequencing and expression studies in patients with MPDs as well cells (18). This includes activation of JAK1, JAK2, Tyk2, STAT1, as AML. STAT2, STAT3, STAT4, and STAT5 (20, 21). IL-27 regulates various aspects of immune responses, including T cell-mediated Materials and Methods immunity (21, 35, 39–42), and can also regulate the activity of Cell Culture and Retrovirus Production. 293T cells were maintained B cells, mast cells, and monocytes (18, 39, 40, 43). Interestingly, in DMEM supplemented with 10% FBS. 32D cells and BaF3 IL-6 can function as a for various cancer cells (44, cells were grown in RPMI medium 1640 supplemented with 10% 45), including AML blast cells (46), suggesting signaling by FBS and 5% WEHI-3B conditioned medium as a source of IL-3. members of this cytokine receptor family can promote oncogenic Ecotropic retrovirus was made in 293T cells by using the pVPack cell growth. In support of this notion, Takeda et al. (47) have system (Stratagene). Stable cell lines were generated by retro- observed hyperproliferation of T cells designed to overexpress viral infection as described in SI Text. IL-27R. In this article, we show that expression of IL-27R induces cDNA Library Construction. Under Institutional Review Board IL-3-independent growth of 32D myeloid and BaF3 pro-B cells approval, AML samples were obtained from the Moffitt Cancer (Figs. 1A and 4A). Because BaF3 cells lack gp130 expression (18, Center Tissue Core Facility as viably frozen mononuclear cells 36), IL-27R-mediated transformation does not depend on gp130. from the bone marrow of untreated patients. mRNA was isolated In addition, we were unable to detect expression of gp130 in 32D by using a FastTrack 2.0 mRNA Isolation Kit (Invitrogen). cells (Fig. 4B), demonstrating IL-27R does not require gp130 to Double-stranded cDNA was prepared with a SuperScript Dou- elicit a transforming signal in myeloid cells. 32D cells trans- ble-Stranded cDNA Synthesis Kit (Invitrogen) and purified in formed by IL-27R have constitutively activated JAK/STAT two fractions of two sizes by using the Geneclean III family members (Fig. 1C), the JAK-binding Box 1 motif of (Q-Biogene). cDNA fractions were ligated into the pEYK3.1 IL-27R is required for its transforming activity (Fig. 3B), and retroviral vector (14), and ligations were transformed into E. JAK inhibition blocks cell transformation by IL-27R (Fig. 2). cloni electrocompetent cells (Lucigen). The library contained These data suggest IL-27R-mediated activation of JAK family Ϸ3.3 million bacterial colonies with a cloning efficiency of members is critical for its transforming capacity. Ϸ90%. JAK family proteins play a major role in myeloid disorders as highlighted by the discovery of the JAK2-V617F point mutation. Screening of cDNA Library and Isolation of cDNA from Cells. 32D cells JAK2-V617F likely contributes to the pathogenesis of various expressing exogenous Bcl2 were infected with retrovirus made MPDs, including polycythemia vera, essential thrombocytosis, from the AML cDNA library. Four independent infections were and myelofibrosis (24–28). Homodimeric type I cytokine recep- done for each cDNA fraction. Two days after infection, cells tors have been shown to be required for JAK2-V617F-mediated were plated in the absence of IL-3 to select for IL-3-independent activation and transformation. Homodimeric receptors, includ- transformants. Genomic DNA was isolated from IL-3- ing EpoR, TpoR, and GCSFR, support the activation of JAK2- independent cells and treated with Cre recombinase (NEB) to V617F in a ligand-independent manner (37). These receptors excise pEYK3.1 plasmids, containing putative transforming likely provide a scaffold upon which mutant JAK2 proteins can cDNAs, which were then isolated by bacterial transformation. interact, which is believed to be necessary for the activation of the mutant kinase. Although IL-27R is a component of a Cell Growth Analysis. To assay 32D and BaF3 cell response to IL-3 heterodimeric cytokine receptor with gp130, we show that it is deprivation, cells were washed twice with RPMI medium 1640/ capable of activating JAK2-V617F in cells in a gp130- 10% FBS. Cells were plated at a concentration of 4 ϫ 105/ml in independent manner (Fig. 5). This finding suggests IL-27R can RPMI medium 1640/10% FBS, and cell growth and viability functionally replace homodimeric cytokine receptors to support were monitored by trypan blue exclusion. the activation of JAK2-V617F. It is still to be determined whether or not IL-27R functions as a homodimer in the context Immunoblot Analyses. Cells were washed in PBS and lysed in lysis of its ligand-independent transforming properties. buffer [25 mM Tris (pH 7.4), 150 mM NaCl, 25 mM NaF, 1% Recently, point mutations in Mpl, the gene for the type I Triton X-100, 1 mM sodium vanadate, 2 mM sodium pyrophos- cytokine receptor TpoR, have been found in JAK2-V617F- phate, 10 ␮g/ml leupeptin, 2 ␮g/ml aprotinin, and 1 mM PMSF]. negative myelofibrosis and essential thrombocythemia patients Protein concentrations were determined with a BCA protein (48, 49). These mutations were identified under the hypothesis assay kit (Pierce Biotechnology), and equal amounts of protein that patients who lack a JAK2 mutation may have other muta- were analyzed by SDS/PAGE. Primary antibodies used in this tions that lead to JAK2 activation, such as mutations in upstream study include: IL-27R (TCCR) (C-term) (Sigma), phospho-(P-) activators of JAK2, including cytokine receptors. The identifi- STAT1(Y701), P-STAT3(Y705), P-JAK1(Y1022/1023), cation of point mutations in Mpl in myeloid disorders suggests P-JAK2(Y1007/1008), P-ERK(T202/Y204), JAK1, JAK2 (Cell mutations in other type I cytokine receptors may also contribute Signaling Technology), P-STAT5(Y694) (BD Transduction Lab- to diseases of the myeloid system. Our data suggest that contri- oratories), and STAT1, STAT3, STAT5, and ERK1 (Santa Cruz bution of heterodimeric cytokine receptors to JAK2-V617F Biotechnology). Immunoblots were developed by using ECL pathogenesis, as well as JAK2-V617F-negative myeloid disor- Western Blotting Substrate (Pierce Biotechnology). ders, should be considered. We suggest that a nonmutated single chain of a heterodimeric Flow Cytometry for IL-27R Expression. Expression of IL-27R was type I cytokine receptor has the ability to transform hemato- determined by flow cytometry using an anti-human TCCR/ poietic cells and that a single component of a heterodimeric type WSX-1 antibody (R&D Systems) labeled with Alexa Fluor-647 I cytokine receptor can functionally replace a homodimeric type (Invitrogen/Molecular Probes). Details are provided in SI Text. I receptor as an activator of JAK2-V617F. In light of these findings, our data suggest that heterodimeric type I cytokine JAK Inhibitor I Studies. 32D cells were washed twice with RPMI receptors may play unappreciated roles in mediating activation medium 1640 containing 0.1% BSA and incubated in the same

18506 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702388104 Pradhan et al. Downloaded by guest on September 28, 2021 medium with either 0.1% DMSO or 0.5 ␮Mor2␮MJAK Box1mt, and MSCVneo-JAK2-WT or V617F (a gift of Ross inhibitor I (EMD Biosciences/Calbiochem) for 3 h before lysis. Levine, Memorial Sloan-Kettering Cancer Center, New York), For cell growth and viability, cells were plated at a concentration and SMARTPool siRNA against gp130 (Dharmacon) or a of 2 ϫ 105/ml in RPMI medium 1640/10% FBS and either 0.1% nonsilencing control siRNA (Qiagen) by using calcium phos- DMSO or 0.5 ␮M JAK inhibitor I. Cell growth and viability were phate precipitation. Cells were lysed and analyzed by immuno- determined by trypan blue exclusion. After 24 h of JAK inhibitor blotting 2 days after transfection. I treatment, the percentage of apoptotic cells and cell cycle profiles were determined by flow cytometry. See SI Text. We thank George Daley for the pEYK3.1 plasmid, Ross Levine for the JAK2-WT and V617F expression plasmids, Amy Hazen for technical Construction of IL-27R Box 1 Mutant. IL-27R Box1 mutant was advice, and William Kerr for helpful discussions. This work was sup- generated by site-directed mutagenesis (Stratagene). See SI ported in part by the Flow Cytometry and Molecular Biology Core Text. Facilities at the H. Lee Moffitt Cancer Center and Research Institute, National Institutes of Health/National Cancer Institute Grant JAK2-V617F Activation Studies. 293T cells were transfected with K01CA098330, the American Cancer Society, and The V Foundation for combinations of pBabe-puro, pBabe-puro-IL-27RWT, or Cancer Research (G.W.R.).

1. Frohling S, Scholl C, Gilliland DG, Levine RL (2005) J Clin Oncol 23:6285– 27. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, 6295. Wlodarska I, Clark JJ, Moore S, et al. (2005) Cancer Cell 7:387–397. 2. Gilliland DG (2001) Curr Opin Hematol 8:189–191. 28. Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, Score J, Seear R, 3. Gilliland DG (2002) Semin Hematol 39:6–11. Chase AJ, Grand FH, et al. (2005) Blood 106:2162–2168. 4. Deguchi K, Gilliland DG (2002) Leukemia 16:740–744. 29. Lee JW, Kim YG, Soung YH, Han KJ, Kim SY, Rhim HS, Min WS, Nam SW, 5. Gilliland DG, Tallman MS (2002) Cancer Cell 1:417–420. Park WS, Lee JY, et al. (2006) Oncogene 25:1434–1436. 6. Carnicer MJ, Nomdedeu JF, Lasa A, Estivill C, Brunet S, Aventin A, Sierra J 30. Levine RL, Loriaux M, Huntly BJ, Loh ML, Beran M, Stoffregen E, Berger (2004) Leuk Res 28:19–23. R, Clark JJ, Willis SG, Nguyen KT, et al. (2005) Blood 106:3377–3379. 7. Reuther GW, Lambert QT, Caligiuri MA, Der CJ (2000) Mol Cell Biol 31. Towatari M, Iida H, Tanimoto M, Iwata H, Hamaguchi M, Saito H (1997) 20:8655–8666. Leukemia 11:479–484. 8. Reuther GW, Lambert QT, Rebhun JF, Caligiuri MA, Quilliam LA, Der CJ 32. Thompson JE, Cubbon RM, Cummings RT, Wicker LS, Frankshun R, (2002) J Biol Chem 277:30508–30514. Cunningham BR, Cameron PM, Meinke PT, Liverton N, Weng Y, et al. (2002) 9. Meyer J, Rhein M, Schiedlmeier B, Kustikova O, Rudolph C, Kamino K, Bioorg Med Chem Lett 12:1219–1223. Neumann T, Yang M, Wahlers A, Fehse B, et al. (2007) Leukemia 21:2171– Nature 2180. 33. Ihle JN (1995) 377:591–594. 10. Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner RJ (1983) 34. Tanner JW, Chen W, Young RL, Longmore GD, Shaw AS (1995) J Biol Chem Proc Natl Acad Sci USA 80:2931–2935. 270:6523–6530. 11. Askew DS, Ashmun RA, Simmons BC, Cleveland JL (1991) Oncogene 6:1915– 35. Takeda A, Hamano S, Yamanaka A, Hanada T, Ishibashi T, Mak TW, 1922. Yoshimura A, Yoshida H (2003) J Immunol 170:4886–4890. 12. Laneuville P, Heisterkamp N, Groffen J (1991) Oncogene 6:275–282. 36. Nandurkar HH, Hilton DJ, Nathan P, Willson T, Nicola N, Begley CG (1996) 13. Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, Asou N, Oncogene 12:585–593. Kuriyama K, Yagasaki F, Shimazaki C, et al. (2001) Blood 97:2434–2439. 37. Lu X, Levine R, Tong W, Wernig G, Pikman Y, Zarnegar S, Gilliland DG, 14. Koh EY, Chen T, Daley GQ (2002) Nucleic Acids Res 30:e142. Lodish H (2005) Proc Natl Acad Sci USA 102:18962–18967. 15. Baffy G, Miyashita T, Williamson JR, Reed JC (1993) J Biol Chem 268:6511– 38. Steensma DP, Dewald GW, Lasho TL, Powell HL, McClure RF, Levine RL, 6519. Gilliland DG, Tefferi A (2005) Blood 106:1207–1209. 16. Nunez G, London L, Hockenbery D, Alexander M, McKearn JP, Korsmeyer 39. Hunter CA (2005) Nat Rev Immunol 5:521–531. SJ (1990) J Immunol 144:3602–3610. 40. Villarino AV, Huang E, Hunter CA (2004) J Immunol 173:715–720. 17. Sprecher CA, Grant FJ, Baumgartner JW, Presnell SR, Schrader SK, Yam- 41. Artis D, Villarino A, Silverman M, He W, Thornton EM, Mu S, Summer S, agiwa T, Whitmore TE, O’Hara PJ, Foster DF (1998) Biochem Biophys Res Covey TM, Huang E, Yoshida H, et al. (2004) J Immunol 173:5626– Commun 246:82–90. 5634. 18. Pflanz S, Hibbert L, Mattson J, Rosales R, Vaisberg E, Bazan JF, Phillips JH, 42. Chen Q, Ghilardi N, Wang H, Baker T, Xie MH, Gurney A, Grewal IS, de McClanahan TK, de Waal Malefyt R, Kastelein RA (2004) J Immunol Sauvage FJ (2000) Nature 407:916–920. 172:2225–2231. 43. Larousserie F, Charlot P, Bardel E, Froger J, Kastelein RA, Devergne O (2006) 19. Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J, Hibbert L, J Immunol 176:5890–5897. Churakova T, Travis M, Vaisberg E, et al. (2002) Immunity 16:779–790. 44. Frassanito MA, Cusmai A, Iodice G, Dammacco F (2001) Blood 97:483–489. 20. Kamiya S, Owaki T, Morishima N, Fukai F, Mizuguchi J, Yoshimoto T (2004) 45. Molnar EL, Hegyesi H, Toth S, Darvas Z, Laszlo V, Szalai C, Falus A (2000) J Immunol 173:3871–3877. Semin Cancer Biol 10:25–28. 21. Lucas S, Ghilardi N, Li J, de Sauvage FJ (2003) Proc Natl Acad Sci USA 100:15047–15052. 46. Saily M, Koistinen P, Zheng A, Savolainen ER (1998) Eur J Haematol 22. Lin TS, Mahajan S, Frank DA (2000) Oncogene 19:2496–2504. 61:190–196.

23. Sternberg DW, Gilliland DG (2004) J Clin Oncol 22:361–371. 47. Takeda A, Hamano S, Shiraishi H, Yoshimura T, Ogata H, Ishii K, Ishibashi CELL BIOLOGY 24. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou T, Yoshimura A, Yoshida H (2005) Int Immunol 17:889–897. GS, Bench AJ, Boyd EM, Curtin N, et al. (2005) Lancet 365:1054–1061. 48. Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M, 25. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, Garcon L, Steensma DP, Elliott MA, Wolanskyj AP, Hogan WJ, et al. (2006) Blood Raslova H, Berger R, Bennaceur-Griscelli A, et al. (2005) Nature 434:1144–1148. 108:3472–3476. 26. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli 49. Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, Cuker A, A, Cazzola M, Skoda RC (2005) N Engl J Med 352:1779–1790. Wernig G, Moore S, Galinsky I, et al. (2006) PLoS Med 3:e270.

Pradhan et al. PNAS ͉ November 20, 2007 ͉ vol. 104 ͉ no. 47 ͉ 18507 Downloaded by guest on September 28, 2021